Methods and compositions including a 13 kD B. burgdorferi protein

ABSTRACT

All  Borrelia burgdorferi  sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osp). Mutants of  B. burgdorferi  lacking Osp proteins were selected with polyclonal or monoclonal antibodies at a frequency of 10 −6  to 10 −5 . One mutant that lacked OspA, B, C and D was further characterized in the present study. It was distinguished from the OspA + B +  cells by its (i) auto-aggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum- and complement-sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in the skin the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two different classes, A and B, of monoclonal antibodies (mAb) with growth inhibiting properties for mutant cells were produced. Class A mAbs bound to 13 kDa surface proteins of  B. burgdorferi  sensu stricto and of  B. afzelii.  The minimum inhibitory concentration of the Fab fragment of one mAb of this class was 0.2 μg/ml. Class B mAbs did not bind by Western Blot to  B. burgdorferi  cells but reacted with cells in an unfixed cell immunofluorescence assay and growth inhibition assay. These studies revealed hitherto unknown functional aspects of Osp proteins, notably serum-resistance, and indicated that in the absence of Osp proteins other antigens are expressed or become accessible at the cell&#39;s surface.

[0001] This application is a C.I.P. of U.S. Ser. No. 08,124,771, filedSep. 21, 1993; whic is a C.I.P. of U.S. Ser. No. 07/781,355 filed Oct.22, 1991, now issued as U.S. Pat. No. 5,246,844.

INTRODUCTION

[0002] Lyme disease is a complex, multisystemic illness caused by atleast three genomic species of the spirochete B. burgdorferi sensu lato(reviewed in ref. 6). virtually all North American isolates have beenclassified as B. burgdorferi sensu stricto (1, 17, 63). Europeanisolates also include two other genomic species, B. garinii and B.afzelii (1, 24). The clinical features and epidemiology of Lyme diseasehave been well characterized (reviewed in ref. 6). Comparatively less,however, is known about the pathogenic features of Lyme diseaseborrerlias and immunopathological responses to them in the host.

[0003] Ignorance of precise mechanisms of Lyme disease pathogenesis ispartly attributable to the paucity of basic information about allspirochetes. The spirochete cell is unique in several aspects (33). Oneof the features of borrelia is the abundance of one or severallipoproteins in the outer cell membrane (16, 19, 20, 34, 43). Much hasbeen learned about immunogenicity, as well as biochemical and geneticaspects, of these lipoproteins in Lyme disease and relapsing feverborrelias (4, 16, 19, 35, 37, 42, 64).

[0004] The lipoproteins OspA and OspB are major contributors toantigenic distinctness of Lyme disease borrelias (6). Both OspA and OspBare co-transcribed from a single operon located on linear plasmid of 49kb in B. burgdorferi sensu stricto (16). Many of European and some NorthAmerican B. burgdorferi sensu lato strains express a thirdimmunodominant major protein, OspC (64). Another protein of this group,OspD, has been also reported (43), Proteins called “OspE” and “OspF”have been reported, but their surface exposure and location in the outermembrane have not been established (39).

[0005] OspA and OspB may contribute to the spirochete's ability toadhere to or invade host cells (15, 26, 62). It has been suggested thatOspA may affect the chemotactic response of human neutrophils in vitro(15). Mitogenic and cytokine-stimulatory properties of OspA and OspBhave been also shown (41). In a previous study we found that reducedsize and amounts of OspB was associated with lowered infectivity (48).The findings of Cadavid et al indicated that differences in invasiveproperties and tissues tropism between serotypes of related spirocheteBorrelia turicatae, a relapsing fever agent, may be determined by theexpression of a single surface protein that is analogous to Osp proteinsof B. burgdorferi (23).

[0006] These studies of function of Osp proteins, however, are stilllimited in number. More information is needed regarding the function ofthese proteins, in particular their roles in infectivity and theircontributions to the microorganism's ability to survive in the host. Oneapproach to obtain these insights is selection and characterization ofmutants with altered surface lipoproteins. There were several compellingreasons for studying B. burgdorferi cells that lacked all known Ospproteins (51, 54). Our first intent was to characterize morphology andfunction of the Osp-less mutant. We asked whether borrelias lackingOspA, B, C, and D would be altered in such functional properties, as (i)generation time, (ii) ability to form colonies on solid medium, (iii)adherence to cells, (iv) serum and complement sensitivity, (v) potentialto evoke immune response after intradermal live cell inoculation, and(vi) ability to survive in the skin. Among pathogenic borrelias the roleof surface lipoproteins in these respects have not yet been reported.

[0007] Another intriguing aspect was the immunological characterizationof the Osp-less mutant. There have been several studies describing lowmolecular weight lipoproteins that have not been identified as Osps.Katona et al showed the presence of a major low-molecular-weightlipoprotein specific for B. burgdorferi and raised the possibility thatit was a borrelial equivalent of Braun's lipoprotein (36). Another studyreported an immunogenic 14 kDa surface protein of B. burgdorferirecognized by sera from Lyme disease patients (55). These findingsencouraged us to determine whether other proteins are present on thesurface in the absence of Osp proteins.

SUMMARY OF INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS Materialsand Methods

[0008] Strains and Culture Conditions

[0009]B. burgdorferi sensu stricto mutants were of the B31 (ATCC 35210)lineage (Table 1). The Osp phenotypes and plasmid contents ofnoninfectious derivatives B311, B312, B313 and B314 were describedpreviously under these or other designations (3, 7, 32, 54). TABLE 1Isolates of B. burgdorferi sensu lato used in the study and their Ospprofile Genomic Osp profile^(a) species Isolate OspA OspB OspC OspDReference B. burgdorferi B31 + + − + 22,54 B311 + + − − 3,7,54B312 + + + − 32,54 B313 − − − − 51,54 B314 − − + − 54 HB19 + + + + 12,60Sh.2 + + + − 57 B. afzelii ACAI + + + − 17 B. garinii IP90 + + + − 1,17

[0010] Populations that were passed in medium not more than 10 timeswere considered low passage isolates. The low passage, infectiousprogenitor for this lineage retained the original strain designation,B31 (22). With the exception of B31. all cells of this lineage weregrown from single cell clones. In some experiments we also used theseother strains (Table 1): HB19 (12, 60) and Sh.2 (57), both which are B.burgdorferi sensu stricto, B. afzelii strain ACAI (17) and B. gariniistrain Ip90 (1, 17). B. hermsii HS1 serotype 33 (ATCC 35209; ref. 11)was abbreviated to Bh33. Borrelias were grown in BSK II medium andharvested by methods described previously (3, 5). When culturing tissuesfrom animals, rifampicin (50 μg/ml), phosphomycin (100 μg/ml) and, forskin samples, additionally amphotericin (25 μg/ml) were added to themedium. Cells were counted in a Petroff-Hauser chamber by phase-contrastmicroscopy. In some experiments borrelias were also grown on solid BSKII medium as described (32, 51). To estimate growth rate, borrelias atan initial concentration of 2×10⁶ cells/ml, were grown in tightlycapped, 13×100-mm polystyrene culture tubes (Falcon Labware, LincolnPark, N.J.) containing 6 ml of medium. Growth at 34° C. in 1% CO₂atmosphere was monitored visually and by cell counts every 12 h for 3 d.The amount of total cellular protein in the final cell pellet wasdetermined with Bradford reagent (Bio-Rad Laboratories, Richmond,Calif., (12). The microscopic aggregation of borrelias alone or in thepresence of antibodies was graded according to the following scale: 0,single cells with less than 10% of the cells in clumps of 2-10 cells;1+, 10-50% of cells in clumps of 2-10; 2+, 10-50% of cells in clumps of11-100; 3+, >50% of cells in clumps of 11-100: and 4+, >50% of cells inclumps of >100.

[0011] Antisera and Monoclonal Antibodies

[0012] The origins of the OspA-specific mAb H5332 (12), OspB-specificmAb H6831 (10) and Vmp33-specific mAb H4825 (10) have been given.Monoclonal antibody H9724 binds to native and denatured flagellins ofdifferent Borrelia species (9). These antibodies are IgG subclass 2a(IgG2a).

[0013] Additional polyclonal and monoclonal antibodies were produced forthis study. Female, 6-8 week old BALB/c mice (Jackson Laboratory, BarHarbor, Me.) were used. Freshly-harvested borrelia were washed with andresuspended in PBS, pH 7.0. The total cellular protein in the suspensionwas estimated with Bradford reagent and adjusted with PBS for a totalprotein concentration of 200 μg/ml, 0.5 ml of antigen suspension wasemulsified in 0.5 ml of complete Freund's adjuvant (CFA; Sigma), and 200μl of emulsion was administered as six subcutaneous injections at day 0.Control mice received a 200 μl emulsion of equal parts of CFA and PBSalone. The total dose per mouse was 20 μg protein. After 4 weeks micewere boosted with the same dose. Mice were bled by eye sinus puncture 10days after the boost. After collection, sera were evaluated by ELISA andGIA. On day 52, the mice received intravenously 2×10⁸ viable borreliasin 100 μl of PBS. Fusion of mouse splenocytes with NS1 myeloma cellswere performed on day 56 by a modification of the method of Oi andHerzenberg (44). Undiluted hybridoma supernatant fluids withoutantibiotics were screened by wet ELISA, unfixed cell IFA and WesternBlot techniques. Those fluids that were positive by either one of thesemethods were then evaluated by GIA. For GIA hybridoma supernatant fluidswere dialyzed against PBS, pH 7.0 and concentrated with Centriprep-10(Amicon) cartridges. The isotypes of antibodes were determined using acommercial kit (Immunotype; Sigma). Ascitic fluids from hybridomas wereproduced as described (51).

[0014] Purified mAbs and univalent Fab fragments were prepared fromhybridoma supernatants essentially as described (53). Briefly, hybridomasupernatants were concentrated using an Amicon 8200 membraneconcentrator with a Diaflo YM30 ultrafiltration membrane (Amicon,Beverly, Ma.) under 50 psi N₂. Purified mAps were obtained by proteinA-sepharose column chromatography. Univalent Fab fragments were preparedusing the Immunopure Fab Preparation kit (Pierce) by cleaving thepurified antibodies with papain, retaining intact immunoglobulin and Fcfragments on a protein A-sepharose column, and dialyzing the void volumeof the column against PBS, pH 7.0. Purified mAbs and Fab fragments wereconcentrated with Centriprep-10 (Amicon). Protein concentrations weredetermined by UV spectrophotometry at 280 nm. Purified whole IgG and Fabfragments were analyzed by SDS-PAGE. Reactivities of purified mAbs andFab fragments were confirmed by direct and indirect immunofluorescenceassay. Western blot and GIA.

[0015] ELISA

[0016] The method for ELISA was essentially as described previously(52). For this “dry” ELISA borrelias at a total protein concentration of1.4 μg/ml in phosphate-buffered saline (PBS), pH 7.0 were dried ontopolystyrene 96-well microtiter plates at 37° C. for 18 h. For a “wet”ELISA borrelias at a total protein concentration of 3 μg/ml in 15 mMNa₂CO₃-35 mM NaHCO₃ buffer, pH 9.6 were coated onto plates at 4° C. for24 h. After blocking for 1 h at 37° C. With 1% (wt/vol) dried nonfatmilk in PBS (milk/PBS) and washing with PBS alone, twofold dilutions ofantibody in milk/PBS were added. The plates were incubated for 2 h at37° C. and washed with PBS. Bound antibody was measured using alkalinephosphatase-conjugated goat anti-mouse IgG (Zymed). The substrate wasp-nitrophenyl phosphate (Sigma). Absorbance values were recorded at 490nm on a model 580 ELISA reader (Dynatech Laboratories, Chantily, Va.);wells with values ≧0.2 were considered positive.

[0017] Immunofluorescence Assays

[0018] Indirect immunofluorescence assay (IFA) of fixed, dried cells wasperformed as described (11, 12). Harvested, fresh borrelias were washedwith RPMI 1640 medium, mixed with a suspension of washed raterythrocytes in 50% RPMI 1640-50% fetal calf serum, and a thin smear ofthe suspenion was coated on the slides. Slides were fixed in methanol,air dried, and kept in a dessicator at −20° C. until use.

[0019] Binding of monoclonal antibodies (mAb) to unfixed livespirochetes was assessed by a modification of the procedure of Barbouret al (12). 10⁷ borrelias were washed with 2% (wt/vol) BSA in PBS/Mg(PBS/Mg/BSA) and then resuspended in 0.5 ml of undiluted hybridomaculture supernatant or 0.5 ml of PBS/Mg/BSA containing the of interest.The cell mature was incubated at room temperature with gentle rotationfor 60 min. The cells were centrifuged, washed twice with PBS/Mg/BSA,resuspended in 30 μl volume of PBS/Mg/BSA with 20 μg/ml of anti-mouseIg-fluorescein F(ab′)₂ fragment (Boehringer-Mannheim, Indianapolis,Ind.) and incubated for 30 min under the same conditions. Beforemicroscopic evaluation the volume of the cell suspension was adjusted to300 μl with PBS/Mg/BSA.

[0020] For direct IFA purified mAbs and their Fab fragments wereconjugated with fluorescein isothiocyanate (QuickTag FITC ConjugationKit; Boehringer-Mannheim). Fractions containing the antibody-fluoresceinconjugate were mixed together, dialyzed in the dark against PBS for 24h, and concentrated with a Centriprep-10 (Amicon, Beverly, Mass.). 10⁷borrelias in log-phase growth were resuspended In RPMI 1640 medium with10-100 μg/ml of antibody-fluorescein conjugate and examined forfluorescence at 3, 15, 30, 60, and 360 min.

[0021] Growth Inhibition Assays

[0022] The growth inhibition assay (GIA) was described previously (53).Briefly, to a 100 μl volume of BSK II containing 2×10⁶ borrelias wasadded an equal volume of heat-inactivated (56° C. for 30 min) mAb orpolyclonal antiserum, serially diluted two-fold in BSK II. To evaluatethe susceptibility of borrelias to fresh, nonimmune serum, we appliedthe same growth inhibition technique using pooled unheated serum fromC3H/HeN mice (Taconic, Germantown, N.Y.). Blood was drawn on ice,separated from red blood cell clot, and immediatelly frozen at −135° C.Heat-inactivated serum from the same mice served as a control. Todetermine the susceptibility of borrelias to complement, unheated orheated (56° C. for 30 min) guinea pig complement (Diamedix, Miami, Fla.)was added to each well at an activity ranging from 6 to 1 hemolytic unit(HU; CH₅₀) per well. In some experiments, 2 HU of unheated guinea pigcomplement were added to each well for a final concentration of 10 HU/mlof medium after addition of antibody. The incubations were performed inflat-bottomed, 96-well, polystyrene microtiter plates, covered byadhesive, clear plastic seals (Sensititre Microbiologic Systems;Westlake, Ohio) and were carded out for 72 h at 34° C. in a 1% CO₂atmosphere. Growth in the wells was monitored visually for changes inthe color of the phenol red indicator and by phase contrast microscopyof wet-mounts of culture samples. A pink color of the indicator afterincubation represented at least 20-fold fewer cells in these wells thanin wells that were yellow. The minimal inhibitory concentration (MIC)was the lowest concentration of mAb that produced pink instead of yellowwells (53). All growth inhibition studies were performed at least twice.

[0023] Electrophoresis and Western Blot Analysis

[0024] Whole-cell lysates were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15% or 17%acrylamide described previously (2, 11). In some experiments, cleavageof surface-exposed proteins of intact borrelias with proteinase K(Boehringer-Mannheim) was carried out (51). For this study 490 μl of asuspension containing 5×10⁸ cells in PBS/Mg was mixed with 10 μl ofproteinase K solution (20 mg/ml of water) and incubated for 40 min at22° C. The reaction was stopped by the addition of phenylmethylsulfonylfluoride. For Western blot analysis, proteins were transferred tonitrocellulose membranes, which were then blocked with 3% (wt/vol) driednonfat milk in 10 mM Tris-HCI (pH 7.4)-150 mM NaCl (milk/TS) for 2 h asdesribed before (44). After a wash in milk/TS, membranes were incubatedwith mAb ascitic fluid diluted 1:50 or 1:100 in milk/TS or hybridomasupernatant fluid diluted 1:5 or 1:10 in milk/TS. Alkalinephosphatase-conjugated recombinant protein A/G (Immunopure; PierceChemical Co., Rockford, Ill.) served as the second ligand. The blotswere developed with nitro-blue tetrazoliumchloride-5-bromo-4-chloro-3-indolylphosphatase ptoluidine salt (Pierce,Rockford, Ill.).

[0025] Adherence Assay

[0026] An assay for adherence of intrinsically-labeled borrelias tohuman umbilical vein endothelium (HUVE) cells was carried outessentially as described (62). Briefly, borrelias were intrinsicallyradiolabeled with [³⁵S]-methionine, washed with PBS and resuspended to adensity of 1.7×10⁸ cells per ml in Medium 199 with 20% fetal calf serum.300 μl aliquots of radiolabeled spirochetes were added to confluent HUVEcell monolayers grown in 24-well plates. After a 4 hr incubation at 4°C., monolayers with associated organisms were washed, solubilized, mixedwith scintillation cocktail (Universol ES; ICN Pharmaceuticals, Irvine,Calif. ), and counted by scintillation. The assay was done withtriplicate samples and performed twice. Differences between borreliapopulations in adhesion were analyzed by

[0027] Student's T Test

[0028] Experiments in Mice

[0029] Six-to-eight week old, female C3H/HeN mice (Taconic, Germantown,N.Y.) were used. Borrelia cells were counted and diluted in BSK II togive the desired inoculum. For live cell immunization, 100 μl of cellsin BSK II medium was transferred to 900 μl of sterile PBS solutionimmediately prior to immunization. 100 μl of this suspension then wasinoculated intradermally in the abdomen at day 0. As a control, 100 μlof 0.1×BSK II in PBS was used. On day 24 mice were bled from the tailvein, and their sera were examined by ELISA and GIA. Mice werechallenged on day 28 at the base of the tail with 10⁴ of B. burgdorferistrain Sh.2 (48). Mice were euthanized 14 d following infection. Plasma(0.5 ml) obtained from citrated blood, the whole bladder, maceratedheart, and cross-cuttings of both tibiotarsal joints were added to BSKII medium and cultured at 34° C. Cultures were examined for the presenceof motile spirochetes by phase-contrast microscopy at days 7 and 14 ofcultivation; they were scored as negative when no motile spirocheteswere seen in forty, 400× fields. For evaluation of borrelia survival inskin, borrelias were diluted in 1×BSK II. The abdominal skin was shaved,and 10⁷ borrelia cells were injected intradermally at 3 or 4 separatelocations. Mice were sacrificed at 0.25, 0.5, 2, 6, 9, 12, 18 and 24 hafter injection and samples of skin from the injection sites wereimmediately cultured in BSK II medium at 34° C.

Results

[0030] Isolate B313 of the B31 lineage of B. burgdorferi lacked OspA, B,C, D (Table 1; 51, 54). This mutant was selected from a clonalpopulation of B311 under the selective pressure of an anti-OspA mAb.Isolate B311's Osp profile was OspA⁺B⁺C⁻D⁻ (54). Mutants that lack bothOspA and OspB were selected with polyclonal or monoclonal antibodiesdirected against B. burgdorferi at a frequency of 10⁶−10⁵ (51). Thegenetic basis for the Osp-less phenotype was loss of a 38 kb and 49 kblinear plasmids and retention of a 16 kb plasmid (43, 51, 54).

[0031] Growth Rate

[0032] Osp-less mutant B313 was easily distinguishable from B311, aswell as from other high-passage, Osp-bearing isolates of the B31lineage, in broth culture by its tendency to form microscopicaggregates. B313 cultures had aggregation scores of 1+ or 2+, whereasB311 had a score of 0. Another observed difference was the decreasedability of B313 cells to turn the phenol red indicator yellow in the BSKII, even when the culture reached stationary phase. One possibleexplanation for this is that metabolic activity of the Osp-less mutantwas lower than that of wild-type borrelias. Alternatively, theOspA⁻OspB⁻ mutant may have a slower rate of growth than its parent B311and, consequently, does not reach the same cell densities as wild-typeborrelias at a particular time point. To examine these possibilities wedetermined the growth rates of B311 and B313 and measured the amount ofborrelia protein in the final cell pellet.

[0033] B311 and B313 cells were grown until stationary phase, that is,when no further growth occurred, was reached. Cell counts weredetermined every 12 h in triplicate, and the log₁₀ of means of cellcounts were plotted against time. At stationary phase B311 cultures hada cell density of 1.5-2.0×10⁸ cells/ml and B313 cultures had a celldensity of 4-5×10⁷ fourfold lower. Protein concentrations in the finalB311 and B313 cell pellets were 0.65 mg and 0.16 mg, respectively, afinding consistent with the cell counts. The mean generation time(±standard error of the mean) of B311 cells was 6.6±0.1 h; the valuesfor B313 cells were 9.5±0.2 h, 50% slower. These findings indicated thatthe Osp-less cells both grew more slowly and achieved a lower final cellmass than did their Osp-bearing counterparts.

[0034] Plating Efficiency

[0035] We also evaluated another biological characteristic of theOsp-less mutant, namely, its ability to grow as a colony on solidmedium. Current procedures for cultivation of different low and highpassage B. burgdorferi on solid medium yield efficiencies of platingbetween 50 and 100% (32, 48, 51). In the previous studies we found wecould plate other antibody-resistant variant populations of the B31lineage with the same high efficiency (51). An exception was the verylow plating efficiency of mutant B314 (Table 1), which lacks all linearplasmids and has an OspA⁻B⁻C⁺D⁻ phenotype (54). These data suggestedthat mutants with Osp⁻ phenotype might also have a lesser ability toform colonies.

[0036] The experiment was performed twice, each time plating intriplicate10¹-10⁶ borrelias per plate. B311 cells grew as colonies withthe expected plating efficiency of 50%. The efficiency of B313 platingwas 0.01%, more than a thousand-fold lower than for B311 cells under thesame conditions. Of three arbitrarily chosen colonies of B311 that grewin broth medium and were then subjected to SDS-PAGE, all retained theOsp-less phenotype (data not shown).

[0037] Adherence to Endothellal Cells

[0038] Adherence of radiolabeled B. burgdorferi B311 and B313 cells toHUVE cell monolayers was measured after 4 h at 4° C. At this temperatureborrelias do not detectably enter endothelial cells and ladherence ofcells becomes maximal by 4 h (27). The assay was repeated twice. Resultsof the two experiments are given in Table 2. The ability of Osp-lesscells to adhere HUVE monolayer both times was only half that ofwild-type borrerlias, a difference that was significant (P<0.001).

[0039] Serum and Complement Sensitivity

[0040] Wild-type B. burgdorferi is resistant to the nonspecificbactericidal activity of nonimmune serum, in spite of classical andalternative complement pathway activation (38). We asked whether theborrelias' ability to resist the nonspecific bactericidal effects ofcomplement might be attributable to Osp proteins. Accordingly, we firstexposed B311 cells and the Osp-less mutant to twofold serially dilutedfresh, naive mouse serum in a GIA Heat-inactivated serum was applied inthe same assay in parallel. As expected, B311 cells were resistant tothe nonimmune serum; no growth inhibitory effect on the cells wasobserved at the lowest serum dilution of 1:8. In contrast, the minimuminhibitory titer of nonimmune serum against Osp-less borrelias was 1:64.In wells with inhibited growth the B313 cells were nonmotile and hadlarge membrane blebs (data not shown). When heat-inactivated TABLE 2Adherence^(b) Mean cpm Cell adhered ± Mean % of inoculum Experiment^(a)population SEM^(c) adhered^(d) I 8311 14719 ± 134 5.0 8313 7360 ± 36 2.5II 8311 13447 ± 92  5.7 8313 8801 ± 83 2.9

[0041] serum was applied to either B311 or B313 cells, growth inhibitionor these morphologic effects were not observed at a serum dilution 1:8.The findings of this experiment suggested that complement affected theOsp-less cells.

[0042] To further evaluate the serum-susceptibility of the Osp-lessmutant, we next compared the effect of different activities of guineapig complement on B311 and B313 cells. The dose of applied complementvaried between 1-6 HU per well, and, as a control, the same doses ofheat-inactivated complement were used. The study was performed twice.Whereas heat-inactivated guinea pig complement had no growth inhibitoryeffect on either isolate at the doses of 6 HU or less per well, therewere substantial differences in the effect of unheated complement onB313 and B311. As little as 1 HU of complement inhibited growth of B313;this represented an MIC of ≦5 HU/ml. The corresponding MIC of unheatedcomplement for B311 cells was ≧25 HU/ml:

[0043] We also estimated the frequency of B313 cells surviving in thepresence of complement. Because of B313's poor growth on solid medium,the experiment was performed in 96-well microtiter plates (51). 5×10⁶ ofB311 or B313 cells were exposed to 3 HU/tube of guinea pig complementfor 6 h. After this time cell suspensions were diluted to theconcentration of complement less than 1 HU/tube and aliquoted in 200 μlvolumes to individual microtiter plate wells at inocula ranging between10⁰-10⁵ cells per well. Cells that were exposed to heat-inactivatedcomplement or no complement at all served as controls. The frequency ofcomplement-resistant mutants of B313 was calculated using tables of thePoisson distribution to be 3-6×10 ⁻⁵.

[0044] Of 11 complement-resistant B313 clones that were transferred tomedium without complement, only 6 proliferated . When these 6 cultureswere again exposed to 3 HU of complement, all were as susceptible as theparent population. This suggested that if some changes had occurred inthe cell, they most likely represented a phenotypic change. When the 6cultures derived from the resistant populations were examined by PAGE,there was no discernable difference between them and the controlB313,protein profiles (data not shown).

[0045] Survival of Borrelias in Skin

[0046] In the previous experiment we showed that outer surfacelipoproteins might have a role in protecting borrelias from onenonspecific host-defense, namely, complement. Borrelias invade the hostthrough the skin, being able to survive in it from a few days to years(59). Accordingly, we next evaluated whether Osp proteins also mightprotect borrelias from nonspecific resistance factors in the skin of themouse, for example, different chemical substances from tissues withantibacterial activity, early inflammation factors, and phagocytic cells(18).

[0047] In a first step assessing these factors, we determined how longB311 and B313 cells would survive in the skin after intradermalinoculation. The experiment was repeated twice. In total 8 to 12separate skin locations were evaluated for spirochetal growth at theeach time point. Mice were sacrificed at 0.25, 0.5, 2, 6,9, 12, 18 and24 h following inoculation, and full-depth skin biopsies were cultured.All cultures from up to 9 h were positive with both B311 and B313. In 12h, 4 out of 8 and 5 out of 8 skin cultures were positive with B311 andB313 cells, respectively. None of the cultures from 18 and 24 h afterinoculation was positive. These findings indicated that OspA and/or OspBmight not benefit the borrelia's survival in the skin. To confirm thatcells that survived in the skin retained the same phenotype, 6 randomlychosen cultures each of B311 and B313 were subjected to SDS-PAGE; all ofthe examined cells retained an unchanged protein profile (data notshown).

[0048] Immunization by Intradermal Inoculation with Live Cells

[0049] The next question addressed was whether rive cells lacking knownOsp lipoproteins were able to induce immune response in the skin and, ifso, how that response differed from the one induced by Osp-bearingcells. A rationale for this experiment was another study, in whichviable but noninfectious B. burgdorferi of strain H819 at singleintradermal dose of 10⁶ live cells per mouse were sufficient protectmice against challenge with 10⁴ Sh.2 cells 4weeks later (49). Thisimmunization dose was used with B311 and B313 cells in the presentstudy. The immune responses of immunized and control mice were evaluatedby ELISA and GIA with B311 and against the challenge strain, Sh.2.

[0050] As shown in Table 3 only immunization with cells expressing OspAand OspB, that is, B311, was effective in protecting all 5 mice fromexperimental infection with 10⁴ cells of the challenge strain. Osp-lessB313 failed to elicit a protective immune response at a immunizationdose of 10⁶ cells. All 5 mice that were immunized with live Osp-lessmutant cells, as well as control mice injected with 0.1×BSK alone,became infected. Immune responses among the groups as evaluated by ELISAand GIA also differed substantially. Whereas B311 cells evoked an immuneresponse as assessed by ELISA and especially by GIA, the response toB313 cells in the same assays was similar to that of the control group.Western blot analysis with sera from mice immunized with B313 showed noresponse to proteins of B. burgdorferi, except for faint bands againstflagellin (52, not shown). Inasmuch as both B313 and B311 appear tosurvive in the skin for the same time span, a possible explanation forthese results of immunization was that Osp proteins are an importantstimulus for the host immune system to recognize the spirochete.

[0051] Polyclonal Antisera to B311 and B313

[0052] The lack of an antibody response to B311 and other Osp-bearingcells by mice immunized with B313 might also be explained by thepresence of unique antigens in B313 cells. According to this hypothesis,antibodies were produced in response to live cell immunization with B313but they were directed against antigens found only in B313 cells. Therehave been reports indicating that B. burgdorferi has other surfaceproteinaceous antigens than those been defined as Osps (19, 36, 40, 55,58). These considerations encouraged us to investigate the possibilityof non-Osp antigens that might still be present on the surface of themutant cells.

[0053] The previous experiment showed that there was little detectableantibody response after live cell Intradermal immunization with Osp-lesscells at a dose that evokes antibodies in animals immunized withOsp-bearing cells. Consequently, to study the immunogenicity of Osp-lesscells-another immunization approach was needed. Mice were immunized withB311 and B313 whole cell emulsified in an adjuvant and boosted once withthe same TABLE 3 Intradermal immunization and protection of mice withlive B311 and B313 Immunogen^(a) Mouse No ELISA^(b) GIA^(c) Experimentalinfection^(d) B311 1 256 1024 2 128 128 3 256 512 0/5 4 256 512 5 128128 B313 1 4 <16 2 8 <16 3 4 <16 5/5 4 4 <16 5 8 <16 Control^(e) 1 4 <162 2 <16 3 4 <16 5/5 4 4 <16 5 4 <16

[0054] preparation. Sera were examined against both immunogens 7 wkafter the initial immunization; the results are presented in Table 4. Wefirst examined the immune response by dry ELISA and found thatreciprocal titers for a homologous reaction were as high as 32,768 Whenheterologous sera were evaluated, the reciprocal titers were still high:16,384 for anti-B311 serum against B313 cells, and 4,096 for anti-B313serum against B311 cells. Sera from mice immunized with CFA alone werenegative at a dilution of 1:2. This experiment confirmed that, besidesknown Osps, there were other immunogenic components recognized by mice.

[0055] Antisera pooled from within the same group were also evaluated byGIA for functional activity (Table 4). To avoid the deleterious effectof complement on Osp-less cells the serum was heat-inactivated. Asexpected, the reciprocal growth inhibitory titer of anti-B311 againstB311 was high at 8,192. Anti-B313 serum did not effect B311 cells at anyof the dilutions examined. Moreover, Osp-less mutant cells wereinhibited by anti-B311 polyclonal serum only at a dilution of 1:32. Thelatter result, while indicating the specificity of the response,nevertheless, suggested that growth inhibitory antibodies to non-Ospcomponents were produced. This was confirmed by examining the Osp-lessmutant cells with homologous anti-B313 serum; the reciprocal growthinhibitory titer was 4,096. There was no growth inhibition of eitherB311 or B313 cells by sera of mice immunized with adjuvant and PBSalone.

[0056] mAbs Against the Osp-less Mutant

[0057] To further characterize the surface antigens of the Osp-lessmutant we produced mAbs to B313. Procedures used for production andscreening of hybridoma supernatant fluids were designed to select forand identify those mAbs that were directed against surface proteins andhad functional activity by GIA. To enhance selection of antibodiesagainst surface components mice were boosted intravenously with liveB313 before the spleen fusion. As a screen for surface-directed mABs, weused an ELISA in which whole borrelias were not dried in the microtiterplate wells. To further evaluate mAbs for surface binding all hybridomasupernatants identified by wet ELISA were examined by unfixed cell TABLE4 Analysis of polyclonal mouse antisera to B311 and B313 cells by ELISAand growth inhibition assay^(a) Polyclonal Mouse ELISA^(b) Growthinhibition assay^(c) serum No B311 B313 B311 B313 Anti-B311 1 1638416384 2 16384 16384 8192 32 3 32768 16384 4 32768 16384 Anti-B313 1 409616384 2 4096 16384 <8 4096 3 4096 32768 4 2048 32768 Control^(d) 1 <4 <4<8 <8 2 <4 <4

[0058] immunofluorescence assay. Using these assays we identifiedseveral mAbs specific for B313 cells.

[0059] Six mAbs produced against the Osp-less mutant were selected forfurther study by Western blot and GIA. We had distinguished twodifferent classes of mAbs, designated A and B, in the screening byunfixed cell IFA. The 3 class A mAbs produced prominent cell blobs and4+ cell aggregates; the 3 class B mAbs produced 3+ aggregates and didnot produce blebs (FIG. 1). The morphologic changes observed with classA mAbs were similar to what was observed by Coleman et al (25) and us(50) when bactericidal antiborelial antibodies were used. Class A mAbswere associated with a homogeneous patchy pattern of binding to wholecells and little fluorescent staining of the background (see below). Incontrast class B mAbs in the wet IFA did not produce staining of singlewhole cells. Instead it was associated with numerous fluorescent spotsin the background (not shown). By GIA class A antibodies were inhibitoryat dilutions of hybridoma supernatant of 1:256-2048; class B mAbsinhibited growth only at dilutions of supernatants of 1:16 or lower.Both class A and B mAbs inhibited the growth of B311 at a dilutions of1:16 or 1:32, but not at higher dilutions. None of the antibodiesinhibited the growth of B. hermsii. When 1 HU of guinea pig complementwas added, it did not increase the inhibitory effect of either class ofmAb against B313 cells.

[0060] Western Blot Analysis of mAbs

[0061] The two classes were also distinguishable by Western Blot Class BmABs did not bind to any protein in the blots, a result that suggestedthese mAbs were directed against conformational epitopes ornon-proteinaceous antigens. In contrast, all class A mAbs were reactiveby Western blot and bound to the same low molecular weight protein. Theresults with two class A mAbs, 15G6 and 7D4, are shown in FIG. 2 Boththese class A mABs were IgG2b. We have already described an OspA⁻OspB⁻B. burgdorferi of HB19 lineage that expressed a surface protein notdetectable in the Osp-bearing wild-type population (51). Therefore, wefirst determined whether other lineages of B31 express the proteinrecognized by 15G6 and 7D4 mAbs. An antibody-reactive protein with anM_(r) of 13,000 was present in all the B31 cell lineages investigatedand in similar amounts. This protein was designated “p13” and was boundby both mAbs. Identically sized proteins bound by 15G6 and 7D4 werepresent in HB19 and Sh2 strains as well (data not shown). Both mAbs alsoproduced minor bands with proteins with with M_(r)'s of 26,000, 32,000,and 44,000 (FIG. 2).

[0062] We next determined if mAbs 15G6 or 7D4 mAbs recognized similar oridentical proteins in other genomic species of Lyme disease borrelias.The results with 15G6 are shown in FIG. 3; the same results wereobtained with 7D4. Representatives of B. afzelii and B. garinii wereevaluated at the same time as B311, B313 and B. hermsii cells by Westernblot The mAb recognized a protein of slightly higher apparent molecularweight in B. afzelii ACAI. Neither 15G6 nor 7D4 recognized any proteinin B. garinii IP90 or B. hermsii.

[0063] We also investigated whether p13 was cleaved from intact cells byproteinase K, as has been shown for other B. burgdorferi surfaceproteins (21). No band was observed by Western blot with either anti-p13kDa mAb after proteinase K digestion of wild-type and Osp-less mutantcells, an indication that p13 is surface-exposed. The result with mAb15G6 and B313 cells is shown in the right panel of FIG. 3.

[0064] Immunofluorescence Studies of p13

[0065] To further assess the topography of p13 in the cell, inparticular to determine if p13 is exposed over B313's entire surface, weused fixed and unfixed cells in indirect (IFA) and direct (DFA)immunofluorescence assays. In this series of experiments we usedpurified 15G6 mAb; for unfixed cell DFA purified 15G6 mAb was conjugatedwith fluorescein.

[0066] In the fixed cell IFA B311 and B313 cells were individually mixedwith a suspension of washed rat erythrocytes and coated as a thin smearover the the slides. No fluorescein-labeled spirochetes were seen witheither wild-type or mutant cells when cells were exposed to 15G6 mAb. Incontrast, anti-flagellin mAb H9724, used as a control, showed uniformfluorescein labelling of fixed to the glass spirochetes, as wasdescribed in ref. 12 This suggested that the epitope for the 15G6 mAbwas sensitive to the experimental conditions and treatment required forthe sample preparation. Although this epitope was accessible to 15G6 mAbby the Western Blot in the wholecell lysates, it was not recognized inthe dried and fixed borrelias.

[0067] We then assessed the binding of fluorescein-labeled antibodies tofixed and unfixed borrelias. B313 cells were examined at 3, 15, 30, 60,and 360 min after addition of the 15Gb6 conjugate. The cells began tofluoresce within 3 min of addition of the conjugate; the antibody wasuniformly distributed over the length of the cell by 30 min (FIG. 4).Cells remained motile for up to 30 min. Cell aggregates and blebs becameevident after 15 min and increased in amounts over the 6 hoursobservation (FIG. 4). In contrast to B313, very few (<1%) of B311 cellswere detectably bound by 15G6 conjugate by DFA with unmixed cells.

[0068] The finding that mAb 15G6 had some inhibitory activity againstB313 cells, albeit only at a low dilution, suggested that p13 of OSA⁺B⁺cells was accessible to some degree to the antibody. We asked whetherthis putative exposure could be increased in wild-type cells by theadditional presence of an anti-Osp antibody in the suspension. Toaddress this question we used either the anti-OspB mAb H16831 oranti-ospA mAb H15332 in combination with the 15G6 conjugate. Bothantibodies of the combination were added at the same time. The 15G6conjugate was also used by itself against B311or B313.Immunofluorescence of cells was examined in 2 h. As expected theconjugate by itself did not bind to B311 cells when used alone (notshown). The conjugate produced aggregation and homogeneous staining ofcells of B313 (FIG. 5). In contrast, the binding of the 15G6 conjugateto B311 cells in the presence of the anti-OspA or anti-OspB mAbs was nothomogeneous. The results with H6831 and the conjugate are shown in FIG.5. The cells were found in large aggregates with patches of fluorescencedispersed throughout the clump of cells. The experiment showed thatsimultaneous exposure to mAbs directed against OspA or OspB resulted inexposure of p13 protein by mAb 15G6. Whether this was directlyattributable to cell lysis or to alterations of the outer membrane andits proteins remains to be determined.

[0069] Functional Characterization of Anti-p13 mAb

[0070] Results of the preceding experiments prompted furtherinvestigation of mAb 15G6 at the functional level. For this we used thewhole purified IgG molecule and univalent Fab fragments of 15G6 mAb. Twobactericidal antibodies, anti-OspB mAb 116831 and anti-Bh33 mAb H4825prepared in the same way served as controls (50). All mAbs were testedwith B311 and B313 cells by GIA (Table 5). As reported previously, theanti-OspB mAb H6831 was highly effective in killing B311 cells but, asexpected, produced no damage on the Osp-less mutant cells. The effect of15G6 mAb to the Osp-less cells, however, was marked. The MIC of thewhole IgG was 20 ng/ml. Univalent fab fragments inhibited growth at aconcentration of 200 ng/ml, the same as was observed with thebactericidal Fab fragment directed against Bh33, H4825 (53). As foundpreviously with hybridoma supernatants, 15G6 in purified form inhibitedgrowth of B311 cells only at 25 μg/ml or above. No growth inhibitionwith either B313 or B311 was observed with the anti-B. Hermosa mAbH4825. This experiment proved the functional importance of the newlyidentified 15G6mAb to the Osp-less mutant cells and provided evidencethat mAbs active as Fab fragments can be produced against other surfaceproteins besides Osp proteins.

[0071] The combination of anti-Osp and anti-p13 mAbs on wild-type cellswas further characterized by GIA. Wild-type cells were exposed totwo-fold serially diluted purified H6831 (anti-OspB) H5332 (anti-OspA),or, as a control, anti-Bh33 mAb H4825. 15G6 mAb was simultaneouslyapplied at 200 ng/ml, 10× the MIC for B313 and less than 0.01× the MICfor B311. GIA without the addition of mAb15G6 was performed in parallel.The results are shown in Table 6. There was no effect with mAb H4825,with or without the addition of 15G6 mAb. There was only a two-folddecrease in the MIC of mAb H5332 when mAb 15G6 was added. The effect ofthe addition of 15G6 to H6831 was more pronounced: without 15G6, itsgrowth inhibitory concentration was 150 ng/ml, whereas with addition of15G6, intensive cell blebbing occured at concentrations 64-128-foldlower, i.e., 1-2 ng/ml. These results were consistent with observationsof the combination by immunofluorescence assay and indicate thatanti-Osp and anti-p13 mAbs are synergistic in their activity againstTABLE 5 Growth Inhibition by purified whole IgG and Fab fragments ofmAbs 15G6, H6831 and H4825 mAbs Minimal growth inhibitory concentration(μg/ml) 15G6 H6831 H4825 Cells Whole IgG Fab fragment Whole IgG Fabfragment Whole IgG Fab fragment B311 12.5 25 0.15 2 >25 >25 B313 0.020.2 >25 >25 >25 >25

[0072] TABLE 6 Growth inhibition by purified whole IgG of mAbs H6831,H5332 and H4825 in combination with mAb 15G6^(a). Minimal growthinhibitory concentration (ng/ml) H6831 H5332 H4825 Without 15G6 150600 >25000 With 15GGa 1-2 300 >25000

[0073]B. burgdorferi

Discussion

[0074] An isolate of B. burgdorferi lacking OspA, B, C, and D wascharacterized with respect to biological functions and its surfaceantigens, in particular a 13 kDa protein. Although we focused on asingle mutant of B. burgdorferi the results are likely also applicableto other strains of B. burgdorferi sensu lato and the other genomicspecies of Lyme disease agents. Other isolates of Lyme disease borreliashave one or more of the Osp proteins (reviewed in ref. 6). The studyshowed that the Osp-less mutant differed in several ways from theOspAB-bearing parent with which it was compared with. Although the mostprominent structural difference between B311 and B313 was their Ospprotein phenotypes, differences in other, less abundant proteins or innon-proteinaceous components may have affected changes in function. Themost apparent genetic difference between the OspA⁺B⁺ B311 and OspA⁻B⁻B313 was the presence or absence of the entire 49 kb linear plasmid.Thus, insights into Osp function from this study will need to be furtherexplored by more direct and specific mutagenesis of these genes.

[0075] Biological characteristics distinguishing Osp-less andOsp-bearing cells was growth rate and the population density at whichstationary phase occurred. Isolate B313 grew more slowly than did B311and stopped dividing at a lower cell density than did B311. This may beattributable wholly or in part to the greater auto-agglutinationdisplayed by the mutant cells. The triad of self-aggregation, slowergrowth rate, and lower cell density at stationary phase have also beennoted with low-passage, infectious isolates of B. burgdorferi (3, 53).Like B313, some low-passage isolates of B. burgdorferi sensu lato alsohave a poor plating efficiency on solid medium (47). The diminishedability of aggregated Osp-less borrelias to move abut the broth mediummay explain their slower growth under that condition, but why B313 cellscould not grow on solid medium when singly dispersed is unknown. Lowplating efficiency also is a feature of B314 cells, which lack the 16 kblinear plasmid as well as the 49 kb plasmid (54). Inasmuch as B314 cellsexpress OspC protein, the lower plating efficiency cannot be attributedto lack of Osp proteins per se.

[0076] Curiously, while OspA⁻B⁻ cells seem to be inherently more stickyfor one another, they were less disposed than OspA⁺B⁺ cells to adhere tohuman endothelial cells. This indicates that the phenomenon ofself-aggregation is not equivalent to the association of the borreliaswith mammalian cells. Prior studies had revealed functions for OspA inendothelial cell adherence and for OspB in cell penetration (26, 28,62). The findings of the present study are also consistent with a rolefor OspA and/or OspB in the association of borrelias with mammaliancells.

[0077] We also examined another possible function of Osp proteins,namely resistance to non-immune effects of serum. For a blood-bornepathogen this would seem to be a requirement for successful transmissionbetween hosts and for proliferation within a mammalian host. Much isknown about what conferes “serum-resistance” to gram-negative and-positive bacteria; less is known about this aspect of spirochetes.Although borrelias have two membranes sandwiching a peptidoglycan layer,as do gram-negative bacteria, the outer membrane of borrelias appears tobe more fluid than that of gram-negative bacteria (8) and lack lipidA-containing glycolipids (61). Thus, it was not likely a priori thatspirochetes would have a similar mechanism for avoiding the alternativecomplements pathway and other non-immune defenses against bacteriaindeed, the results suggest that OspA andfor OspB protect the cells fromcomplement attack. When OspA, B, C, and D are lacking, the borreliaswere more susceptible than OspA⁺B⁺ cells to unheated, nonimmune serumand to guinea pig complement.

[0078] Whatever protection OspA and OspB appeared to confer to theborrelias in serum did not seem to provide an advantage to cells inskin. In this experiment we used two isolates that are not infectious bythe criterion of detectable dissemination to the blood or other tissues.Still, we expected that the Osp-bearing cells would survive for a longerperiod in the skin than would their Osp-less counterparts. This did notoccur in either of the experiments in which this was examined. By 18hours after inoculation both B311 and B313 could not be recovered fromskin samples placed in culture medium. Infectious isolates persist inthe skin for days (14, 47). The limited duration of survival noted inthe present study may also be a function of inherent strain differences.A non-infectious isolate of strain HB19 of B. burgdorferi survived inthe skin for 24 hours by the same culture criterion (49).

[0079] Given the indistinguishability of B311 and B313 with respect toskin survival, one might expect that the immune responses to intradermalinoculation of viable borrelias would be comparable. Although theOsp-less mutant lacked two proteins, OspA and OspB, that areimmunodominant when syringe inocula of 10⁵ or greater are used (13, 29,30, 46, 56), other antigens, such as flagellin, commonly recognized byantibodies in immune sera were still present. Instead we found thatthere was little detectable immune response to B. burgdorferi by ELISA,GIA, and infectious challenge when B313 was the immunogen. Under thesame conditions and with the same dose, mice given B311 had high titersto B. burgdorferi by immunoassays and were protected against challengewith strain Sh.2. The experiment's results suggest that OspA and/or OspBnot only are immunodominant antigens but also, perhaps through theirmitogenic properties (41), immunostimulatory.

[0080] This experiment also raised the possibility that there were noantigens on the cell surface in B313 cells. Without Osp proteins, thecell surface of B. burgdorferi conceivably could be like Treponemapallidum's outer membrane, which is notably inert to the immune system(45). To further assess this we immunized mice with B313 but this timeused adjuvant to enhance immune responsiveness. When this was done, theantiserum produced to B313 cells inhibited the growth of homologouscells but only minimally that of B311. The similar ELISA titers for bothanti-B311 and anti-B313 sera against homologous and heterologous cellsindicated that with the appropriate adjuvant B313 could elicitantibodies to antigens shared with B311. The GIA results showed thatthere were unique features of the surface of B313 cells. Thesecomponents were either not expressed by B311 cells or were otherwisecloaked in these cells. The minimal effectiveness of polyclonalanti-B311 sera in inhibiting the growth of B313 cells indicated thatantibodies to OspA andlor OspB conferred growth inhibition.

[0081] The remaining antigens of the Osp-less mutant were furtherinvestigated with mAbs. The screening procedures were designed toidentify antibodies that had the functional activity of growthinhibition. The antibodies selected by this means fell into two classes:one in which the antibodies in broth medium produced large aggregatesand prominent membrane blebs and a second in which the antibodiesproduced smaller aggregates and minimal evidence of lysis. The firstantibodies were found to bind to a 13 kDa (p13) protein in Westernblots. The second group of antibodies did not bind to any component inblots. For the remainder of this study p13 and mAbs to it werecharacterized in more detail.

[0082] The evidence that the 13 kDa protein was surface-exposed in theOsp-less mutant was the following: (i) agglutination of viable cells byantibody; (ii) growth inhibition by whole immunoglobulin and Fabfragment; (iii) direct immunofluorescent staining of live cells by anantibody conjugate; and (iv) cleavage of antibody's epitope from thecell's surface by in situ treatment with protease. p13 was present inall members of the B31 lineage and in approximately equal amounts. Theexpression of the protein did not vary according the amount of one oranother the Osp proteins. A slightly larger protein recognized by themAb was present in a B. afzelii strain. If Ip90 a representative of B.garinii, have a homologous protein it does not share the mAbs' epitope.

[0083] We considered whether p13 was Identical to one of the other lowmolecular weight B. burgdorferi proteins to which antibodies have beendeveloped. Like antibody to p13, antibody to a 10 kDa protein, asreported by Katona et al. (36), bound to only a small proportion ofOsp-bearing cells in immunofluorescence assays. However, the molecularsize of 10 kDa protein did not vary between strains and uniformfluorescein labeling was seen in fixed cell preparation when probed withmAb to 10 kDa protein (36). Furthermore, 15G6 does not bind to the 10kDa in Western blots (31). Sambri et al. reported the presence of a 14kDa protein of B. burgdorferi (55). This was identified with a mAb andby immunofluorescence of live borrelias. In contrast with what wasobserved by us with mAbs to p13 and by Katona et al. with antibody tothe 10 kDa protein (31). antibody to the 14 kDa protein of Sambri et al.bound to the majority of cells (48). These differences suggest that p13is neither the 10 kDa nor 14 kDa proteins of B. burgdorferi.

[0084] The effect of 15G6 on susceptible borrelias was similar to whatwas observed with the anti-OspB mAb H6831 (50). Binding to the cells wasdetectable by direct immunofluorescence by 3 minutes, The staining washomogeneous and was followed by the appearance of membrane blobs andfurther cell aggregation even with Fab fragments. The concentration of15G6 mAb at which growth inhibition and cell disruption occurred was 20ng/ml. This was 10fold lower to what was observed with H6831 mAb againstB. burgdorferi and the same as with H4825 against B. hermsii (53).

[0085] The failure of mAbs to p13 to inhibit the growth of Osp-bearingcells is consistent with lack of surface exposure of the protein, or atleast impairment of the antibody's access to its target. The cloaking orobstruction could be from OspA, OspB, or a complex of the two. It wasalso possible that p13 was not in the outer membrane at all in B311cells; in those cells it may have been in the periplasmic space or inthe cytoplasmic membrane. Evidence against this latter possibility was(i) cleavage of anti-p13 mAbs epitopes from Osp-bearing cells by in situtreatment with proteinase K, (ii) the finding that when mAb 15G6 to p13was added to antibodies to either OspA or OspB, the growth inhibitoryconcentration for the anti-Osp antibodies was decreased, substantiallywith the bactericidal H6831 to OspB. My itself mAb 15G6 had nodiscernible effect against B311 cells except at high concentrations. Thefinding suggested that p13 was exposed to mAb 15G6 when antibodies toOspA or OspB gathered together Osp proteins in patches in the fluidouter membrane (12). The immunofluorescence provided visual evidence ofthis; large membrane blebs of B311 cells treated with anti-OspA or -OspBproteins were bound by conjugated 15G6 mAb. This in vitro synergismbetween the two antibodies, one directed against an Osp protein and theother against p13. suggests to us that p13 in combination with OspA orOspB could be useful for immunoprophylaxis against Lyme disease.

[0086] The results also lead to other questions about the interaction ofantibodies and borrelias, in particular those lacking the known Ospproteins. The target or targets for the second class of mAbs remains tobe determined. It is also possible that they also bind to p13 but thattheir epitopes are sufficiently conformation-dependent that Westernblots would be negative. Alternatively, there may be other proteins orother non-proteinaceous components in the outer membrane against whichthese functional antibodies act.

[0087] The work was supported by the grants AI-29731 to A.G.B. andAI-26804 D.D.T. from the National Institutes of Allergy and InfectiousDiseases, therefore the United States Government owns rights to thepresent invention.

BRIEF DESCRIPTION OF DRAWINGS

[0088]FIG. 1. Phase contrast photomicrograph of aggregation of B.burgdorferi B313 by monoclonal antibodies. A (upper) panel, 4+aggregation (see Methods) by class A antibody. B (lower) panel, 3+aggregation by class B antibody. Bar, 1.0 μm.

[0089]FIG. 2. Coomassie blue-stained polyacrylamide gel (CB) and Westernblot analysis (WB) of whole-cell lysates of B. burgdorferi isolates B31,B311, B312, B313, and B314 with either monoclonal antibody 15G6 and 7D4.The acrylamide concentration was 17%. The molecular size standards(×1000) were ovalbumin (44), carbonic anhydrase (29), β-lactoglobulin(18), lysozyme (14), and bovine trypsin inhibitor (5.6).

[0090]FIG. 3. Western blot analysis with antibody 15G6. Left panel, B.burgdorferi B311 and B313, B. afzefii ACAI, B. garnii IP90 and B.hermsii Bh33 were probed with the antibody 15G6 mAb. Right panel, B313cells treated (+) or untreated (−) with proteinase K (PK). The molecularsize standards (×1000) were carbonic anhydrase (29), β-lactoglobulin(18), lysozyme (14), and bovine trypsin inhibitor (5.6).

[0091]FIG. 4. Binding of flourescein-conjugated monoclonal antibody 15G6 to B. burgorferi B313. Left-upper and right-upper panels, directimmunofluorescence of unfixed cells in suspension for 3 min (left) and15 min (right). Lower panel, phase contrast photomicrograph ofaggregates and membrane blebs (arrow head). Bar, 10 μm.

[0092]FIG. 5. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels, B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm, FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

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[0157] The above references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, areall specifically incorporated herein by reference.

What is claimed is:
 1. A protein composition, free from total cellcomponents, the protein being characterized as having a molecular weightof about 13 kD, as determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS/PAGE), and being isolatable from B.burgdorferi.
 2. The protein composition of claim 1, wherein thecomposition further comprises B. burgdorferi outer membrane proteinsOspA, OspB, OspC or OspD, in combination with a pharmacologicallyacceptable diluent or carrier.
 3. A purified protein having thefollowing characteristics: (a) being isolatable from B. burgdorferi; (b)being present on the surface of B. burgdorferi cells that lack the outermembrane proteins OspA, OspB, OspC and OspD; (c) being sensitive tocleavage with proteinase K; (d) having a molecular weight of about 13kD, as determined by SDS/PAGE; (e) having binding affinity for themonoclonal antibodies termed 15G6 and 7D4.
 4. The purified protein ofclaim 3, further defined as being isolated from B. burgdorferi cells. 5.The purified protein of claim 3, further defined as being a recombinantprotein obtained from a recombinant host cell that includes a nucleicacid segment that expresses said protein.
 6. The purified protein ofclaim 3, in combination with a pharmacologically acceptable diluent orcarrier.
 7. The purified protein of claim 3, linked to a detectablelabel, the label being a radioactive label, a flourogenic label, anuclear magnetic spin resonance label, biotin or an enzyme thatgenerates a colored product upon contact with a chromogenic substrate.8. An antibody that has binding affinity for the protein of claim
 3. 9.The antibody of claim 8, linked to a detectable label, the label being aradioactive label, a flourogenic label, a nuclear magnetic spinresonance label, biotin or an enzyme that generates a colored productupon contact with a chromogenic substrate.
 10. The antibody of claim 9,linked to an alkaline phosphatase, hydrogen peroxidase or glucoseoxidase enzyme.
 11. The antibody of claim 8, further defined as amonoclonal antibody.
 12. The antibody of claim 11, further defined asthe monoclonal antibody 15G6 or 7D4.
 13. A method for detecting B.burgdorferi in a sample, comprising contacting a sample suspected ofcontaining B. burgdorferi with a first antibody in accordance with claim8, under conditions effective to allow the formation of immunecomplexes, and detecting the immune complexes so formed.
 14. The methodof claim 13, wherein the first antibody is the monoclonal antibody 15G6or 7D4.
 15. The method of claim 13, wherein the first antibody is linkedto a detectable label and the immune complexes are detected by detectingthe presence of the label.
 16. The method of claim 13, wherein theimmune complexes are detected by means of a second antibody linked to adetectable label, the second antibody having binding affinity for saidfirst antibody.
 17. The method of claim 13, further defined as a methodof diagnosing Lyme Disease, wherein the sample suspected of containingB. burgdorferi is a clinical sample obtained from a patient suspected ofhaving Lyme Disease and the detection of immune complexes is indicativeof a patient with Lyme Disease.
 18. A method for detecting antibodies toB. burgdorferi, comprising contacting a sample suspected of containingantibodies to B. burgdorferi with a protein in accordance with claim 3,under conditions effective to allow the formation of immune complexes,and detecting the immune complexes so formed.
 19. The method of claim18, wherein said protein is linked to a detectable label and the immunecomplexes are detected by detecting the presence of the label.
 20. Themethod of claim 18, wherein the immune complexes are detected by meansof a second antibody linked to a detectable label, the second antibodyhaving binding affinity for said protein.
 21. The method of claim 18,wherein the immune complexes are detected by means of a second antibodylinked to a detectable label, the second antibody having bindingaffinity for the first, anti-B. burgdorferi antibodies.
 22. The methodof claim 18, further defined as a method of diagnosing Lyme Disease,wherein the sample suspected of containing antibodies to B. burgdorferiis a clinical sample obtained from a patient suspected of having LymeDisease and the detection of immune complexes is indicative of a patientwith Lyme Disease.
 23. An immunodetection kit comprising, in suitablecontainer means, a protein in accordance with claim 3 or a firstantibody in accordance with claim 8, and an immunodetection reagent. 24.The immunodetection kit of claim 23, wherein the immunodetection reagentis a detectable label that is linked to said protein or said firstantibody.
 25. The immunodetection kit of claim 23, wherein theimmunodetection reagent is a detectable label that is linked to a secondantibody that has binding affinity for said protein or said firstantibody.
 26. The immunodetection kit of claim 23, wherein theimmunodetection reagent is a detectable label that is linked to a secondantibody that has binding affinity for a human antibody.
 27. A method ofgenerating an immune response, comprising administering to an animal apharmaceutically acceptable composition comprising an immunologicallyeffective amount of a protein that has a molecular weight of about 13kD, as determined by SDS/PAGE, and is isolatable from B. burgdorferi.28. The method of claim 27, wherein the composition further comprises aB. burgdorferi OspA, OspB, OspC or OspD protein.
 29. The method of claim27, wherein the 13 kD protein is a recombinant protein.
 30. A mutant B.burgdorferi that lacks the OspA, OspB, OspC and OspD proteins.